Ribosomal DNA is deceptively simple in concept and notoriously difficult in practice. In every animal cell, the genes encoding the 18S, 5.8S and 28S ribosomal RNAs sit together in a cluster, separated by internal transcribed spacers and flanked by external transcribed spacers. This cluster is repeated in long tandem arrays at the nucleolar organizer regions of one or more chromosome pairs, and these arrays are transcribed by RNA polymerase I into a single precursor molecule that is subsequently cut into the three mature rRNAs. Between the repeat units lies the intergenic spacer, a non-coding stretch that carries the promoter and terminator signals controlling pre-rRNA synthesis, along with enhancer elements, replication origins and replication fork barriers that keep the transcription and replication machineries from colliding. While the rRNA genes themselves are among the most conserved sequences in the genome, the spacers evolve rapidly through substitutions, insertions and duplications, which makes them valuable markers for molecular systematics and species identification.

The technical obstacle that has kept most avian rDNA out of reach is its chemical composition. In birds, the transcribed spacers are extraordinarily rich in guanine and cytosine, and the intergenic spacer is built from complex high-order repeats. Conventional short-read sequencing technologies, whether Sanger or next-generation platforms, simply cannot generate reads long enough to span these repetitive, GC-dense regions, and targeted sequencing with site-specific primers is practically impossible even with reagents designed for GC-rich templates. The chicken rDNA sequence was only obtained in 2019 by applying Pacific Biosciences long-read sequencing to a bacterial artificial chromosome clone carrying an rDNA insert. The Japanese quail team took a different long-read route, using Oxford Nanopore Technology on a MinION device to read the quail genome in fragments long enough to cross the entire repeat unit.

The researchers added a clever twist to overcome the stickiness of the target molecules. Because DNA fragments enriched in GC pairs and internal repeats tend to form complex secondary structures and intermolecular connections, the team used a barcoded adapter from a rapid barcoding kit to dilute the GC-rich quail DNA to one tenth of the flow cell load, mixing it with non-targeted DNA of lower GC content. Although this reduced overall coverage, it improved the proportion of reads with complex secondary structure that could be successfully sequenced. From 36,524 raw nanopore reads, 74 aligned to the 18S to 28S rDNA region, and these were assembled into 13 contigs, one of which reached 19,964 base pairs and contained partial external transcribed spacer sequences plus the complete set of rRNA genes. A single read of 21,166 base pairs ultimately covered the entire intergenic spacer, allowing the team to close the gap that had defeated previous attempts.

Validation came from independent sources. The team first mined publicly available Illumina data from the NCBI database, selecting 510 reads that aligned to conserved chicken rDNA sequences and assembling them into three contigs overlapping the 18S to 28S region. Comparison between the nanopore assembly and these Illumina contigs showed similarities of 88.7 percent for the 5-prime ETS to ITS1 region, 90.3 percent for ITS1, and 88.4 percent for the 28S gene, confirming the quality of the consensus. The final rRNA gene sequences were then corrected against the Illumina data and checked with GATK Haplotype Caller algorithms. Cross-checking against raw reads from an alternative sequencing project maintained by the French National Institute for Agriculture, Food, and Environment showed homology of up to 90.6 percent and coverage of up to 99 percent. The complete sequence was deposited in GenBank under accession OK523374.

The architecture of the quail repeat unit mirrors the general plan established in the chicken but with telling differences. The 21,166-base-pair unit comprises a 5-prime external transcribed spacer of 1,779 base pairs, the 18S gene of 1,823 base pairs, ITS1 of 2,047 base pairs, the 5.8S gene of 157 base pairs, ITS2 of 658 base pairs, the 28S gene of 4,185 base pairs, a 3-prime external transcribed spacer of 639 base pairs, and an intergenic spacer of 9,878 base pairs. The coding genes proved highly conserved relative to chicken: 99.5 percent identity for 18S, complete identity for 5.8S, and 85.7 percent for 28S. The two species, which belong to the same family Phasianidae and diverged roughly 35 million years ago, share the same chromosome number and show no interchromosomal rearrangements, making the differences in their spacers all the more informative about the pace of rDNA evolution.

Those differences are concentrated in the non-coding portions. Every spacer region of the quail rDNA carries a slightly lower GC content than its chicken counterpart: 74.1 percent versus 74.9 percent in the 5-prime ETS, 72.1 percent versus 79.4 percent in the 3-prime ETS, 76.8 percent versus 82.1 percent in ITS1, 78.0 percent versus 82.2 percent in ITS2, and 66.3 percent versus 69.2 percent in the intergenic spacer. The quail ITS1 is 401 base pairs shorter than the chicken version, and ITS2 is 76 nucleotides shorter. More striking is the abundance of bendable DNA elements. The quail spacers are saturated with polypurine and polypyrimidine tracts capable of forming non-canonical secondary structures, including the H-form of DNA, and the repeat element GAGGGG may promote the formation of G-quadruplexes. Dispersed inverted repeats such as (CGG)92 and (GCC)78 in the 5-prime ETS, along with an array of microsatellites throughout ITS1, ITS2 and the IGS, suggest that these sequences can adopt sticky, structurally unusual conformations in the cell.

The biological significance of these bendable elements may lie in the peculiar karyotype of the Japanese quail itself. Unlike most birds, which carry a single pair of nucleolar organizer chromosomes, the quail has three pairs. One sits on the microchromosome orthologous to chicken chromosome 16, near the major histocompatibility complex, while the other two are terminal organizers on the short arms of unidentified acrocentric microchromosomes. Remarkably, these two microchromosomes are absent from the current chromosome-level genome assembly of the quail, with no molecular markers or defined gene linkage groups described for them. The authors suggest that the bendable, sticky DNA in the spacers may play a role in nucleolus formation by bringing together nucleolar organizers from different chromosomes, and that transposons previously implicated in relocating rRNA gene clusters to microchromosome termini may explain why the clusters themselves show no significant sequence differences between chromosomal locations.

Beyond comparative genomics, the work touches on questions of genome stability and cell physiology. Ribosomal DNA copy number and conformation influence chromatin stability, sensitivity to damage and the pace of cellular aging, and inactive rDNA copies can sequester proteins unconnected to ribosome biogenesis. The human rDNA repeat unit, the best studied of any species, spans about 43 kilobases in 100 to 500 copies per cell and contains terminator sites, pseudogenes and binding sites for regulatory proteins. Building a comparable reference for every major taxonomic group, the authors argue, is essential for understanding both the conserved and species-specific aspects of nucleolar biology. For birds, the quail sequence now provides the second anchor point, and the team hopes that continued advances in long-read sequencing will eventually pin down the two elusive GC-rich microchromosomes and complete the picture of how avian ribosomal DNA evolves across the genome.

Subject of Research: Complete structure of the ribosomal DNA repeat unit in the Japanese quail genome

Article Title: Description of the complete rDNA repeat unit structure of Coturnix japonica Temminck et Schlegel, 1849 ( Aves )

Article References: Zhukova, A., Zakharov, G., Pavlova, O., & Saifitdinova, A. (2024). Description of the complete rDNA repeat unit structure of Coturnix japonica Temminck et Schlegel, 1849 (Aves). Comparative Cytogenetics, 18, 183-198. https://doi.org/10.3897/compcytogen.18.127373

Image Credits: AI Generated

DOI: 10.3897/compcytogen.18.127373

Keywords: ribosomal DNA, Japanese quail, Coturnix japonica, intergenic spacer, Oxford Nanopore sequencing, nucleolar organizer, comparative cytogenetics, GC-rich sequences, genome assembly, avian genomics, transcribed spacers, chromosome evolution

R2 retrotransposons are non-long-terminal-repeat retroelements with a remarkable lifestyle. Rather than scattering randomly across the genome like many transposable elements, R2 elements insert with exquisite precision into a conserved site within the 28S ribosomal RNA gene cluster. They accomplish this through a mechanism called target-primed reverse transcription. The R2 protein recognizes and cleaves the target DNA site, and the exposed DNA end then primes reverse transcription of an RNA template carried by the element itself. Because the RNA donor both encodes the protein machinery and serves as the template for integration, the entire system can in principle operate with RNA as the only genetic material delivered into a cell, a property that has earned the approach the label all-RNA-mediated DNA integration.

The appeal of such a system for genome editing is considerable. Viral vectors, including lentiviruses and adeno-associated viruses, can insert therapeutic genes but do so with limited control over where the DNA lands, raising the risk of insertional mutagenesis. CRISPR-associated technologies, by contrast, typically require a double-strand DNA break at the target site, which can trigger chromosomal rearrangements, p53-mediated toxicity and unpredictable repair outcomes. R2-based integration sidesteps both problems: it makes no double-strand break and its natural target specificity confines insertion to the repetitive ribosomal DNA array, a locus that has been proposed as a genomic safe harbor because thousands of copies exist and disruption of individual copies is well tolerated.

The new study builds on earlier work from the same laboratory. In 2024, the team reported in Cell that a rationally engineered R2 retrotransposon could mediate targeted gene integration in mammalian cells using only RNA components. That first-generation system, however, was derived from a limited set of source elements, and its efficiency in clinically relevant primary cells remained modest. Subsequent structural and biochemical studies by other groups, including cryo-electron microscopy analyses of R2 complexes captured during target-primed reverse transcription, revealed how the protein and its RNA cofactors cooperate to nick, unwind and copy DNA, providing a mechanistic foundation for rational engineering.

To widen the search space, the researchers turned to birds. R2 elements are widespread across animal lineages, but avian genomes had been comparatively underexplored. The team downloaded and screened 1,139 avian genomes using a computational pipeline built on sequence homology searches, clustering tools such as CD-HIT, protein domain annotation with InterProScan, and multiple sequence alignment with MAFFT followed by maximum-likelihood phylogenetic reconstruction with IQ-TREE. From this survey they identified 159 full-length avian R2 retrotransposons, distributed across multiple bird orders, and systematically catalogued which protein regions and RNA untranslated-region elements were conserved across the family and which varied.

That comparative map proved essential for engineering. Focusing on an element designated R2Tg, the team dissected two previously under-characterized protein regions. Region 1, located near the amino terminus, turned out to be indispensable: truncating it sharply reduced integration efficiency, and the defect could be rescued by fusing the chromatin-remodeling factor HMGN1 or by substituting domains such as nucleosome-binding or chromatin-unfolding modules. Region 2, comprising spacer segments and N-terminal extensions, modulated activity in a length-dependent manner. The analysis also clarified the roles of nuclear localization signals and showed that Region 1 must act in cis with the rest of the protein, since supplying it in trans failed to restore function.

The RNA donor itself proved equally amenable to optimization. The researchers varied the length of the right homology arm, the poly(A) tail, and the 5′ untranslated region, finding that a short four-nucleotide right homology arm sufficed when paired with an appropriate poly(A) tract and an intact 5′ UTR. Covariation modeling across the 165 avian R2 5′ ribozymes revealed a conserved J1/2 secondary structure whose disruption by heterologous substitutions reduced integration, confirming that the ribozyme fold, not merely its sequence, is functionally important. A conserved buffer region in the 3′ UTR was likewise characterized for length and nucleotide composition. These donor-design rules distinguish the new toolkit from earlier R2 systems and contributed to marked gains in both integration efficiency and the proportion of full-length, intact insertions.

Whole-genome sequencing of edited cells confirmed high on-target specificity, with insertions concentrated in the ribosomal DNA array and minimal off-target activity. Long-term culture experiments showed that integrated transgenes, including a GFP reporter, maintained stable expression over months, and that copy numbers remained constant. The team also examined the fate of the integrated cassette, documenting DNA methylation dynamics at the CMV promoter and testing promoter and orientation configurations that maximize durable expression. Notably, re-delivery of R2 protein mRNA induced only negligible indel formation at ribosomal DNA loci, below 0.1 percent, and did not erode transgene expression, suggesting the system can be re-dosed without progressive damage to the target locus.

The most clinically significant results came in primary human cells. In primary T cells, the researchers screened viral accessory proteins for their ability to enhance integration and identified Vp4, the ORF4a protein from Middle East respiratory syndrome coronavirus, which suppresses antiviral stress responses. Co-delivery of Vp4 with an engineered R2 element designated R2SPs substantially improved integration while preserving cell viability and expansion. The team went on to integrate a full CD19 chimeric antigen receptor cassette into primary T cells, generating CAR-T cells that were positive for the receptor by day two and demonstrated cytotoxic activity against target tumor cells in coculture assays. Parallel experiments in primary natural killer cells and primary human foreskin fibroblasts showed that the same tool achieved robust, stable integration across cell types that are notoriously difficult to engineer.

The authors report efficiencies reaching 60 percent site-specific integration across human primary cells, a figure that, if reproduced across laboratories, would place RNA-only gene writing among the most efficient nonviral integration methods available. The sequencing data have been deposited in the Genome Sequence Archive for Human under accession HRA013312, and the analysis code is publicly available on GitHub, lowering the barrier for other groups to adopt and extend the resource. Several of the authors have filed patent applications related to the technology, signaling commercial interest in R2-based gene writing.

Challenges remain before such systems reach the clinic. The ribosomal DNA target, while abundant and tolerant of insertion, constrains applications to loci where expression from the rDNA array is adequate, and controlling transgene copy number and expression level will require further tuning. Delivery of large RNA molecules into primary cells still relies on electroporation, and the immunogenicity of both the R2 protein and coronavirus-derived accessory factors in vivo is untested. Nevertheless, by mining evolution’s own library of site-specific integrases and applying systematic protein and RNA engineering, the study demonstrates that retroelements long studied as genomic fossils can be refashioned into programmable tools, opening a path toward gene insertion therapies that require no virus, no DNA break and no DNA cargo at all.

Subject of Research: Engineering avian R2 retrotransposons for RNA-mediated targeted DNA integration in human cells

Article Title: Discovery and engineering of avian R2 retrotransposons for all-RNA-mediated targeted DNA integration in human cells

Article References: Chen, Y., Hu, Y., Luo, S., Mao, B., Tang, N., Wang, C., Wang, Y., Bai, H., Wang, X., Peng, C., Chen, Q., Xiao, J., Zou, Y., Wang, P., Niu, R., Zhang, J., Zhao, Y., Liang, C., Wang, Y., … Li, W. (2026). Discovery and engineering of avian R2 retrotransposons for all-RNA-mediated targeted DNA integration in human cells. Nature Biotechnology. https://doi.org/10.1038/s41587-026-03315-w

Image Credits: AI Generated

DOI: 10.1038/s41587-026-03315-w

Keywords: R2 retrotransposon, gene integration, genome editing, retroelement, avian genomics, target-primed reverse transcription, CAR-T cells, ribosomal DNA, nonviral vector, RNA donor, primary T cells, Nature Biotechnology